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目的利用慢病毒载体构建稳定过表达人谷氨酰半胱氨酸连接酶催化亚基(GCLC)的WRL68细胞株。方法采用PCR方法合成GCLC基因全长,经Not I、Nsi I酶切后克隆到慢病毒载体LV6上;采用PCR、酶切、测序鉴定重组质粒LV6-GCLC;将重组质粒转染293T细胞,收集培养上清并感染WRL68细胞,经嘌呤霉素筛选出稳定过表达细胞株。采用增强型绿色荧光蛋白检测转染情况,采用Real-time PCR及Western blotting鉴定所构建的细胞株;MTT法检测细胞活性;采用酶联免疫吸附(ELISA)法检测细胞谷胱甘肽(GSH)含量。结果 PCR、酶切及测序结果表明重组慢病毒载体构建成功;重组慢病毒转染293T细胞后可观察到荧光及蛋白表达,包装过表达慢病毒并检测其浓缩滴度为1.0×109 TU/ml;用1μg/ml嘌呤霉素成功筛选出稳定过表达细胞株;GCLC过表达组中GCLC的m RNA和蛋白的表达量高于空载体转染组及对照组(P<0.05);GCLC过表达组、空载体转染组及对照组的细胞活性间比较,差异无统计学意义(P>0.05);GCLC过表达组GSH含量明显高于空载体转染组及对照组(P<0.05)。结论成功构建了稳定过表达人GCLC的WRL68细胞株。
Objective To construct a WRL68 cell line stably overexpressing human glutamylcysteine ligase catalytic subunit (GCLC) using lentiviral vector. Methods The full-length GCLC gene was synthesized by PCR and cloned into lentiviral vector LV6 after cleavage with Not I and Nsi I. The recombinant plasmid LV6-GCLC was identified by PCR, restriction enzyme digestion and sequencing. The recombinant plasmid was transfected into 293T cells and harvested. The supernatant was cultured and infected with WRL68 cells, and a stable overexpression cell line was selected by puromycin. The transfected cells were detected by enhanced green fluorescence protein (GFP). The cell lines were identified by Real-time PCR and Western blotting. The cell viability was measured by MTT assay. The levels of glutathione (GSH) content. Results The results of PCR, restriction enzyme digestion and sequencing showed that the recombinant lentiviral vector was constructed successfully. Fluorescence and protein expression were observed after 293T cells were transfected with recombinant lentivirus. The recombinant lentivirus was overexpressed and its concentration titer was 1.0 × 109 TU / ml ; The stable overexpression cell line was screened with 1μg / ml puromycin. The expression of m RNA and protein in GCLC overexpression group was higher than that in empty vector transfected group and control group (P <0.05); GCLC overexpression (P> 0.05). The GSH level in GCLC overexpression group was significantly higher than that in empty vector transfected group and control group (P <0.05). Conclusion WRL68 cells stably over-expressing human GCLC were successfully constructed.